2.4.2 DEFINICIÓN DE SENSOR
2.4.2.2 CLASIFICACION DE LOS SENSORES
Abstract
It is recognized that microorganisms inhabiting natural sediments significantly mediate the erosive response of the bed (“ecosystem engineers”) through the secretion of naturally adhesive organic material (EPS: extracellular polymeric substances). However, the relative importance of the different EPS producers on the stabilization of the sediment matrix is still unknown. The aim of the first experiment was to examine the adhesive capacity of mono-species biofilm surfaces of benthic diatoms and cyanobacteria, as well to find out whether the combination of two or three species in a biofilm would lead to any kind of additive or synergistic effect on
the adhesive force. Three species, Navicula hansenii, Amphora coffeaeformis and
Oscillatoria species, were grown separately or combined on non-cohesive artificial sediment. The adhesive capacity of the biofilm produced by these species was measured by MagPI over a two week experimental period and related to biological data from Chapter 4. In the present experiment the adhesive properties of the biofilm of the three species (ANO) produced continuously higher adhesive values during the experiment than other biofilms. These results support the main hypothesis and quite clearly point in the direction of an increasing level of adhesive force with increasing
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level of biodiversity of the biofilms. It was the first attempt to explore earlier unknown ground and increased knowledge of the area of species contribution in biofilms. This study was made possible by using a high resolution experimental set-up Magnetic Particle Induction (MagPI). This knowledge will lead to a deeper understanding of the effect of changing biodiversity on interspecies relationships and related implications for the properties and quality of biofilms.
Since the natural ”microalgal mats” is certainly not devoid of heterotrophic bacteria, the question of the functional role and origin of EPS in microbial mats requires further interpretation and can initially be addressed by separate studies of the engineering potential of prokaryotic and eukaryotic assemblages. The aim of the second experiment was to investigate microbial biostabilisation capacity by using natural benthic bacteria and microalgae cultures growing on artificial sediments over 4 weeks. The sediment stability was measured using both a Cohesive Strength Meter (CSM) and a newly developed device Magnetic Particle Induction (MagPI). The results obtained suggest that stabilisation was significantly higher for the bacterial assemblages (up to a factor of 2) than for axenic microalgal assemblages. The EPS concentration and the EPS composition (Chapter 4) were both important in determining stabilisation. The peak of engineering effect was significantly greater in the mixed assemblage as compared to the bacterial (x1.2) and axenic diatom (x1.7) cultures. The possibility of synergistic effects between the bacterial and algal cultures in terms of stability was examined and rejected although the concentration of EPS did show a synergistic elevation in mixed culture. The rapid development and overall stabilisation potential of the various assemblages was impressive (x7.5 and x9.5, for MagPI and CSM, respectively, as compared to controls). This study confirmed the important role of heterotrophic bacteria in “biostabilisation” and highlights the interactions between autotrophic and heterotrophic biofilm consortia.
5.1.Introduction
In intertidal habitats, the cohesive strength of sediments depends on their physicochemical properties such as water content, density, mineralogy, plasticity, salinity and pH (Dade et al. 1992). Benthic communities colonize these habitats and
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form pronounced biofilms (de Winder et al. 1999) which can have a large impact on the whole sediment system. The initial step of biofilm formation is normally regarded as the attachment of microbial cells to a surface by the secretion of polymeric substances. In transient biofilms, however, much of the extracellular polymeric substances (EPS) are secreted as a by-product of the locomotive mechanism of diatoms (Consalvey et al. 2004). In recent years it has been shown that benthic biofilms can also act as a protective layer at the sediment surface that can significantly influence erosion and deposition of sediment particles (Underwood and Paterson 2003). Thus investigation into this “biostabilisation” process is very important in terms of the prediction of sediment erosion potential (Perkins et al. 2004). The major mechanism of this microbial biostabilisation is through the production of EPS matrix which is a complex mixture of carbohydrates, proteins and proteoglycans, secreted by biofilms cells. Previous studies on the influence of EPS on sediment stability have been carried out both in the laboratory (Dade et al. 1992, Battin et al. 2003, Droppo et al. 2007) and in the field (Tolhurst et al. 2000, Hirst et al. 2003) using artificially
modified sediment (Droppo 2001) and/or natural sediment (Underwood and Smith
1998, Yallop et al. 2000, Perkins et al. 2003, Gerbersdorf et al. 2005). However biological impact is highly variable and difficult to express as one constant factor. Numerous studies have established a positive correlation between sediment stabilization, EPS and microbial biomass. Some studies have attempted to use
chlorophyll a/microalgae biomass as an indicator of sediment stability, but the
relationships were at best site-specific (e.g. Riethmueller et al. 2000, Defew et al. 2002, Le Hir et al. 2007). However, although biostabilisation has been increasingly studied over the last decade, there are still significant gaps in our knowledge.
Motile epipelic diatoms are recognized as the main EPS producers in intertidal muddy sediments and as the main contributors to biostabilisation (de Brouwer et al. 2005). MPB alter sediment properties (e.g. erodibility) both directly, by forming a mat on the sediment surface, and indirectly by modifying the activities of benthic infauna (Miller et al. 1996). Nevertheless, due to the microalgal influence on the structure and behaviour of sedimentary habitats, they have been put forward as important “ecosystem engineers” (Boogert et al. 2006), irrespective of their small size that is easily compensated by biomass. The adhesion/cohesion mechanism with the EPS matrix the closely related to the biological function of the polymer in nature has been
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discussed (Hu et al. 2003). For instance, for most benthic cyanobacteria, the adhesion mechanism with the matrix is due to the surface hydrophobicity of exopolymers (Fattom and Shilo 1984). The biological function of EPS production and characterisation from benthic algae and cyanobacteria were described in detail by Parikh & Madamwar (2006) who suggest that cyanobacterial EPS is composed of a network of macromolecules having different biochemical properties, which may contribute to extracellular functions (Kawaguchi and Decho 2000). Due to different degrees of substitution and different structures of the main chains, EPS producers were characterized as strong or weak species in terms of cohesion stabilization, nevertheless their EPS were similar both in protein content, in monosaccharides composition and linkage types (Hu et al. 2003). However there is still a significant gap in the knowledge of engineering capacity of microalgae species and their individual contributions to the biostabilisation processes. There is evidence that MPB is highly sensitive to changes of environmental conditions and depends from a range of abiotic factor, such as salinity, temperature, UV radiations and presence of pollutants (Dejong and Admiraal 1984). These changes in environmental conditions may have a direct impact on MPB community structure as one species dominates (out competes) another or a species disappears/collapse population. In this context, the knowledge about the contribution of species to biostabilisation is very important. The first part of this study will make a first attempt to resolve the contribution to the adhesion from
individual species. The adhesive capacity of the two benthic diatom, Navicula hansenii
(N), Amphora coffeaeformis (A) and Oscillatoria species (O) was examined over two week of experimental period by MagPI. These results were related to EPS (spectrophotometric determination of carbohydrates and proteins) and diatom
biomass (spectrophotometric determination of chlorophyll a) described in previous
chapter (see 4.3.1). A further purpose was to test the hypothesis that higher diversity would lead to increased surface adhesion, because most biofilms found in nature show a higher biodiversity than laboratory systems and that the conjunction of all those species may give some advantages for the biofilm and the system.
While biostabilisation by microalgae has been researched extensively in the marine habitat, the ubiquitous heterotrophic bacteria have largely been ignored, even in conceptual models. However, heterotrophic bacteria also secrete copious amounts of EPS and may have a significant influence in the stabilization of sediment (Dade et al.
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1990, Gerbersdorf et al. 2008). Pioneering studies on the entrainment of a clay-water suspension by Dade et al. (1996) and on the stability of experimentally derived biofilms by Leon-Morales et al. (2007) indicate significant effects of bacterial exopolymers on the substratum. In recent works (Gerbersdorf et al. 2008, Gerbersdorf et al. 2009) it has been shown that natural benthic bacterial assemblages from estuarine areas significantly stabilized test substratum, exceeding by far the importance that might be assumed form the dearth of the literature. Despite their importance in marine ecosystems, marine bacteria and their interaction with
microalgae are rarely studied in this context(Ribalet et al. 2007).
The aim of the second part of this study was to examine the individual engineering capability of the main biofilm components (heterotrophic bacterial and autotrophic microalgae) in terms of their relative functional contribution to substratum stabilisation. It was hypothesized that the coexistence of bacteria and microalgae will show synergistic effects on their engineering potential to enhance EPS production and stabilize the substratum. For this purpose, stabilisation potential of bacterial assemblages (B), axenic autotrophic microalgal/diatom assemblages (D) and mixed assemblages of both (BD) growing on non-cohesive glass beads were determined over a period of 25 days. The adhesive capacity as well as the cohesive forces, both proxies for sediment stability, were monitored regularly by MagPI and CSM, respectively, and related to microbial growth: bacterial cell numbers, bacterial dividing rate, microalgal biomass and EPS secretion: concentrations/composition of carbohydrates and proteins (described in Chapter 4).
5.2.Experimental set-up
Experiments were performed as described in detail in Chapter 4 (see 4.2.1 and 4.2.2). Briefly, for the first experiment of investigation stabilisation capacity of axenic
microalgae culture (4.2.1) the microalgae culture of Navicula hansenii, Amphora
coffeaeformis and Oscillatoria species were obtained from monospecific laboratory
cultures. A layer of 0.5 cm of <63 µm glass beads in total was placed in disposable
plastic trays (70Lx70Wx25H in mm) and 50 ml of autoclaved seawater were added in each box. The control (C) contained only glass beads and autoclaved seawater and was treated regularly by the mixture of antibiotics, to prevent bacterial contaminations. Five replicates were established for each treatment and the
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treatment names refer to the first letter(s) of inoculated culture: A for Amphora
coffeaeformis, N for Navicula hansenii and O for Oscillatoria species. The adhesive capacity of the microalgae biofilm was monitored regularly by MagPI (2.13.2), over the experimental period of two weeks (measured on days 1, 4, 7, 12, 15).
The second experiment investigates the engineering effects on a non-cohesive test bed as the surface was colonised by natural benthic assemblages (prokaryotic, eukaryotic and mixed cultures). The bacteria and microalgae culture were isolated from natural sediment (as described 2.2.1 and 2.2.2) and were grown both separately and simultaneously on a non-cohesive artificial substratum (Ballotini balls, glass beads). A three cm layer (minimum operation depth of the Cohesive Strength Meter,
CSM) of <63 µm glass beads was placed in Rotilab deep-freezes boxes
(208Lx208Wx94H in mm) and 2 L of autoclaved seawater were carefully added to each box. Six replicates per treatment were established and treatment names refer to the first letter(s) of the corresponding culture inoculated: B for bacteria, D for diatoms and BD for the mixed culture of bacteria and diatoms. The controls (C) containing only glass beads and seawater were regularly treated with a mixture of antibiotics. The adhesive capacity and the cohesive forces, both proxies for sediment stability, as determined by MagPI (2.13.2) and CSM (2.13.1) respectively, were monitored regularly over the experimental period 7 times in 4 weeks.
5.2.1. Statistics
The data violated assumptions of normality and homogeneity of variance (visual assessment of the frequency histogram and normal plot, Kolmogorov-Smirnov and Barlett tests), thus differences between treatments were assessed using a non- parametric Kruskal-Wallis (χ²) test (KW), followed by the non-parametric Student- Newman-Keuls (SNK) test to correct for multiple comparisons. Additionally, the Mann-Whitney test was used occasionally to compare pairs of treatments.
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5.3.Results
5.3.1. Investigation stabilisation potential of axenic microalgae culture using
Navicula hansenii, Amphora coffeaeformis and Oscillatoria species
The stability of the substratum
The stability of the sediment surface increased continuously in most treatments up to day 12 (Figure 5.1) and decreased from day 12 until the end of the experimental period. In contrast, there were no significant changes in sediment adhesion/stability for control C sediment, for which the adhesion measurements did not exceed 5 mTesla (to increase the contrast between treatments these data are not presented here). The increase was more pronounced for treatments O and ANO (Figure 5.1 A, Table 5.1).
Table 5.1:Differences between the minimum (the first of sampling day) and maximum
values reached, as well as differences between mixed assemblages ANO and the given treatments (A, N, O, AN, AO, NO, ANO) both times expressed as quotient/factors for MagPI.
Factor Treatment MagPI
Between min and max values
A 1 N 0.9 O 1.2 AN 1.1 AO 1.0 NO 1.1 ANO 1.2
Between ANO and single and combined treatments A 1.4 N 1.3 O 1.2 AN 1.3 AO 1.1 NO 1.2
Statistical testing revealed that the differences between the treatments were significant. For example on day 12, stability was significantly different in all
treatments (KW, χ2=30.37, df= 6, p<0.001). The stability of the biofilm produced by
ANO was significantly higher than all treatments ((up to 1.4 times, MW test, U=0, p<0.001), Figure 5.1, Table5.1). Pair-combined treatment stability was not as high as
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the ANO treatment stability but higher than that for the single species cultures, for instance on day 12, treatment AO was significantly higher than treatment A (Mann- Whitney (MW) test, U=0, p<0.001) and N (MW test, U=2, p<0.05). Single treatment O was significantly higher than treatment A and N (MW test, U=2, p<0.05) (Figure 5.1 A).
Figure 5.1: Mean values (n=5 per treatment, ± SE) of MagPI measurements over the
course of the experiment. (A) The different treatments were single culture: ▲- Amphora;
◊ -Navicula;
●
- Oscillatoria and their mixture:□
- Amphora + Navicula + Oscillatoria. (B) Pairs of mixed cultures:∆ - Amphora + Navicula, ○
- Amphora+ Oscillatoria;♦
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At the day 12, adhesion properties of the biofilm in treatments A, N and AN declined as compared to the first day of the experiment (Figure 5.2 A). Cumulative stability during experimental period was more pronounced for group AN, AO, and ANO (up to 27 mTesla) followed by NO>A>N>O (Figure 5.2 B). The adhesive capacity of the mixed culture biofilm ANO was 5.7 times higher than the control and the single culture was 4.7 times higher than the control (Figure 5.2 B).
Figure 5.2: Adhesion capacity as measured by MagPI: (A) between the first sampling
day and day 12th where most of the variables showed their maximum value. (B)
Cumulative adhesion values (n=25) during 2 weeks of experiment. The treatment name (Diatom species) was given according to the first letter of the corresponding culture(s) inoculated: A for Amphora, N for Navicula, O for Oscillatoria and their mixture AN for Amphora and Navicula, AO for Amphora and Oscillatoria, NO for Navicula and Oscillatoria and ANO for Amphora, Navicula and Oscillatoria.
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Relationship between biological variables (described in Chapter 4) and surface adhesion/ stability
There was a strong positive relationship between adhesion capacity as measured by
MagPI and chlorophyll a (r=0.508, N=35, p<0.01) and colloidal carbohydrate
concentration (r=0.492, N=35, p<0.01) (Figure 5.3 A and B respectively), positive but not significant correlation was found between sediment stability and colloidal proteins concentrations (r=0.145, N=35, p>0.05).
Figure 5.3: Relationship between adhesion capacity as measured by MagPI (mTesla)
and biological variables (n=35). MagPI versus chlorophyll a concentrations (A) and MagPI versus colloidal carbohydrates concentrations (B).
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5.3.2. The stabilisation potential of individual and mixed assemblages of natural
bacteria and microalgae
The stability of the substratum
The surface adhesion of the substratum, as determined by MagPI, increased for all treatments over time to a maximum value on day 14 (Figure 5.4 A, Table 5.2).
Table 5.2: Differences between the first sampling day 1 and day 14 where most of the
variables showed their maximum value, as well as differences between the given treatments (mixed: BD, Bacteria B, Diatom D); both times expressed as quotient/factors for MagPI and CSM.
Factors MagPI CSM between day 1-14 B 3.4 4.0 D 2.6 2.8 BD 2.9 1.8 between treatments BD/B 1.4 2.6 BD/D 2.5 4.1 B/D 1.7 1.3
Cohesion of the substratum as indicated by CSM increased continuously for all treatments (Figure 5.4 B, Table 5.2) over the 4 weeks. The control treatments (C) did not show any significant changes in adhesion/stability over the 25 d of the experiment. There was a significant difference in the means of the treatments for the surface adhesion and cohesion (p<0.05) for all dates except at the beginning of experiment. The mixed assemblage (BD) showed the highest surface adhesion of the sediment followed by the bacterial culture (B) and finally, the diatom biofilms (D). The CSM measurements confirmed the MagPI results with significantly higher sediment surface stability in treatment BD followed by B and D (for example, day 24: KW,
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Figure 5.4: Mean values of sediment stability over the course of the experiment: A by
MagPI (n=6, ±SE) and B by CSM (n=6, ±SE). The different treatments were bacteria and diatoms (BD, ▲), diatoms (D, ♦), bacteria (B, □) and controls (C, ●).
There was a strong linear relationship between CSM (erosion threshold) and MagPI (surface adhesion) (Pearson correlation coefficient: r=0.785, n=20, p<0.001, Figure 5.5).
CHAPTER 5. Coexistence of organisms: sediment stability 122
y = 14.215x + 2.328
R
2= 0.617
0
5
10
15
20
25
0
0.2
0.4
0.6
0.8
1
CSM (Nm
-2)
M
a
g
P
I
(m
T
e
s
la
)
Figure 5.5:The linear relationship between MagPI (mTesla) versus CSM (Nm-2).
In order to visualize possible additive/synergistic effects of bacteria-diatom assemblages for sediment stability, their absolute value of adhesion was compared to the values for the pure bacterial and diatom cultures ([BD]-[B+D], Figure 5.6 A and B). There was a stronger case for interference in the mixed assemblage since the results were much lower than would be expected from the additive effects of the two cultures B and D, as was particularly evident for surface adhesion as determined by MagPI (Figure 5.6 A and B).
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Figure 5.6: The relative assessment between treatments for sediment stability as
measured by MagPI (A) and CSM (B). Substratum stability by the mixed BD treatment relative to the stability of the single B and D treatments is given for MagPI (A) and CSM (B). Where the stability created by the mixed culture (BD) exceeds that of the added single cultures (B and D), the value is positive (synergistic effect) and vice versa (inhibitory effect). If the added values of the single cultures equals the mixed cultures then the effect measured is additive.
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Relationship between biological variables and surface adhesion/ stability
The data of sediment stability measurement (MagPI and CSM) was addressed to
biological variables (see 4.3.2 Chapter 4): chlorophyll a concentration, bacterial cell
number and EPS concentrations. There was a strong positive relationship between
sediment stability measurements and chlorophyll a concentrations (MagPI: r=0.395,
p<0.001; CSM: r=0.501, p<0.001). Similarly, colloidal carbohydrate concentrations were highly significantly correlated with MagPI and CSM measurements for all treatments (Figure 5.7 A and C, Table 5.3). The same applied for the relationship of colloidal protein concentrations to adhesion (MagPI) and cohesion (CSM) of the surface for B and BD, while for D the relationships were not significant (Figure 5.7 B and D, Table 5.3).
Table 5.3: Pearson’s correlation coefficients between surface adhesion (MagPI) and
substratum stability (CSM) and colloidal carbohydrates and proteins per treatment. The significance levels are the following: *** p < 0.001. ** p< 0.01. * p < 0.05.
Treatments
Techniques Carbohydrates Proteins
Diatom MagPI 0.882 17 *** -0.189 21
CSM 0.869 11 *** 0.321 15
Bacteria MagPI 0.861 15 *** 0.770 14 **
CSM 0.753 9 * 0.902 10 ***
Bacteria + Diatom MagPI 0.706 15 ** 0.741 15 **
CHAPTER 5. Coexistence of organisms: sediment stability 125 M a g P I (m T e s la ) C S M ( N m -2 ) Carbohydrates (µg cm -3) Proteins (µg cm-3) y = 0.1117x + 5.5308 R² = 0.516 0 5 10 15 20 25